Tissue transglutaminase (TGase) is an approximately 80 kDa protein that exhibits GTP-binding and hydrolytic activities like signaling G proteins, as well as an enzymatic (transamidation) activity that catalyzes covalent linkages between glutamine residues and primary amino groups, leading to the formation of new protein-protein and protein-polyamine complexes. TGase has been implicated in a number of important biological responses including neuronal development and degeneration, as well as cellular differentiation and apoptosis. During the past funding period, we have found that TGase plays a key role in retinoic acid (RA)-induced differentiation by maintaining cell viability and protecting against apoptotic signals. Both the GTP-binding and transamidation activities of TGase, and its interactions with the retinoblastoma protein (Rb), all appear to be linked to its survival activity. We now propose that TGase-mediated cell survival may be an important outcome of a number of different extracellular stimuli and cell signaling pathways. The studies outlined in this renewal application will set out to directly test these ideas and further our understanding of the molecular mechanisms underlying TGase-mediated cell survival and the intricate regulation of this interesting protein. Three main experimental aims are proposed. 1.) Establish a role for the GTP-binding/GTP hydrolytic activities of TGase in cell survival. These studies will take advantage of our recently determined X-ray crystal structure for GDP-bound TGase to introduce mutations that perturb specific steps in the GTP-binding/GTP hydrolytic cycle and thus can serve as new dominant-active and dominant-negative TGase mutants in cellular studies. 2.) Establish a role for Rb in TGase-mediated survival. The importance of the stable binding of TGase to Rb, as well as the transamidation of Rb, for TGase-mediated cell survival and different cellular functions of Rb will be determined. 3,) Delineation of signaling pathways leading to the up-regulation of TGase expression. Here we will establish the general role that TGase activity plays in cell survival and in the regulation of apoptotic programs by examining the signaling pathways that lead from the nerve growth factor (NGF) receptor as well as from 13-amyloid to TGase expression in rat pheochromocytoma (PC12) cells. By better understanding the mechanisms of action and regulation of this important GTP-binding protein/acyl transferase, we expect to gain fundamental information regarding the balance between cell growth and differentiation versus apoptosis.